This invention relates to the fields of specific binding pair interactions, bioentity separations and the isolation of rare substances from biological fluids. Methods are provided for enhancing such bioseparations, preferably via enhanced magnetic loading onto target entities, thereby facilitating biochemical and diagnostic analysis of target entities so isolated.
There are a substantial number of manufacturing, analytical and laboratory processes and procedures which involve specific binding pair interactions. Many laboratory and clinical procedures are based on such interactions, referred to as bio-specific affinity reactions. Such reactions are commonly utilized in diagnostic testing of biological samples, or for the separation of a wide range of target substances, especially biological entities such as cells, viruses, proteins, nucleic acids and the like. It is important in practice to perform the specific binding pair interactions as quickly and efficiently as possible. These reactions depend on classical chemical considerations such as temperature, concentration and affinity of specific binding pair members for one another. In the ideal, separations employing specific binding partners which rapidly form multiple non-covalent bonds are utilized. The use of such binding partners is important, particularly when the concentration of one of the specific binding pair members to be isolated is extremely low, as often is the case in biological systems. Of course, concentration considerations are relevant in other separation processes, such as in water purification, or in applications where it is necessary to remove trace contaminants or other undesirable products.
Various methods are available for binding, separating or analyzing the target substances mentioned above based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of the resulting complexes from solution or from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the ligand substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation, may be employed for this purpose. However, processes relying on magnetic principles are preferred.
Magnetic particles generally fall into two broad categories. The first category includes particles that are permanently magnetizable, or ferromagnetic; and the second comprises particles that demonstrate bulk magnetic behavior only when subjected to a magnetic field. The latter are referred to as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, materials exhibiting bulk ferromagnetic properties, e.g., magnetic iron oxide, may be characterized as superparamagnetic when provided in crystals of about 30 nm or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter due to strong particle-particle interaction.
Magnetic particles can be classified as large (1.5 to about 50 microns), small (0.7-1.5 microns), and colloidal or nanoparticles ( less than 200 nm). The latter are also called ferrofluids or ferrofluid-like particles and have many of the properties of classical ferrofluids. Liberti et al pp 777-790, E. Pelezzetti (ed) xe2x80x9cFine Particle Science and Technology, Kluver Acad. Publishers, Netherlands,
Small magnetic particles are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents.
In addition to the small magnetic particles mentioned above, there is a class of large magnetic particles ( greater than 1.5 microns to about 50 microns) which also have superparamagnetic behavior. Such materials include those invented by Ugelstad (U.S. Pat. No. 4,654,267) and manufactured by Dynal, (Oslo, Norway). Polymer particles are synthesized, and through a process of particle swelling, magnetite crystals are embedded therein. Other materials in the same size range are prepared by performing the synthesis of the particle in the presence of dispersed magnetite crystals. This results in the trapping of magnetite crystals thus making the materials magnetic. In both cases, the resultant particles have superparamagnetic behavior, readily dispersing upon removal of the magnetic field. Unlike magnetic colloids or nanoparticles referred to above, such materials, as well as small magnetic particles, because of the mass of magnetic material per particle are readily separated with simple laboratory magnetics. Thus, separations are effected in gradients as low as a few hundred gauss/cm, to up to about 1.5 kilogauss/cm. Colloidal magnetic particles (below approximately 200 nm) require substantially higher magnetic gradients for separation because of their diffusion energy, small magnetic mass/particle and stokes drag.
U.S. Pat. No. 4,795,698 to Owen et al. relates to polymer-coated, sub-micron size colloidal superparamagnetic particles. The ""698 patent describes the manufacture of such particles by precipitation of a magnetic species in the presence of a biofunctional polymer. The structure of the resulting particles, referred to herein as single-shot particles, has been found to be a micro-agglomerate in which one or more ferromagnetic crystallites having a diameter of 5-10 nm are embedded within a polymer body having a diameter on the order of 50 nm. These particles exhibit an appreciable tendency not to separate from aqueous suspensions for observation periods as long as several months. Molday (U.S. Pat. No. 4,452,773) describe a material which is similar in properties to those described in the ""698 patent of Owen et al. produced by forming magnetite and other iron oxides from Fe+2/Fe+3 via base addition in the presence of very high concentrations of dextran. Materials so produced have colloidal properties. This process has been commercialized by Miltenyi Biotec, (Bergisch Gladbach, Germany). Those products have proved to be very useful in cell separation assays.
Another method for producing superparamagnetic colloidal particles is described in U.S. Pat. No. 5,597,531. In contrast to the particles described in the ""698 patent, these latter particles are produced by directly coating a biofunctional polymer onto pre-formed superparamagnetic crystals which have been dispersed by sonic energy into quasi-stable crystalline clusters ranging from about 25 to 120 nm. The resulting particles, referred to herein as direct-coated or DC particles, exhibit a significantly larger magnetic moment than the nanoparticles of Owen et al. or Molday et al. having the same overall size.
Magnetic separation techniques utilize magnetic field generating aparatus to separate ferromagnetic bodies from the fluid medium. In contrast, the tendency of colloidal superparamagnetic particles to remain in suspension, in conjunction with their relatively weak magnetic responsiveness, requires the use of high-gradient magnetic separation (HGMS) techniques in order to separate such particles from a fluid medium in which they are suspended. In HGMS systems, the gradient of the magnetic field, i.e., the spatial derivative, exerts a greater influence upon the behavior of the suspended particles than is exerted by the strength of the field at a given point.
High gradient. magnetic separation is useful for separating a wide variety of biological materials, including eukaryotic and prokaryotic cells, viruses, nucleic acids, proteins, and carbohydrates. in methods known heretofore, biological material has been separable by means of HGMS if it possesses at least one characteristic determinant capable of being specifically recognized by and bound to a binding agent, such as an antibody, antibody fragment, specific binding protein (e.g., protein A, streptavidin), lectin, and the like.
HGMS systems can be divided into two broad categories. One such category includes magnetic separation systems that employ a magnetic circuit that is situated externally to a separation chamber or vessel. Examples of such external separators (or open field gradient separators) are described in U.S. Pat. No. 5,186,827. In several of the embodiments described in the ""827 patent, the requisite magnetic field gradient is produced by positioning permanent magnets around the periphery of a non-magnetic container such that the like poles of the magnets are in a field-opposing configuration. The extent of the magnetic field gradient within the test medium that may be obtained in such a system is limited by the strength of the magnets and the separation distance between the magnets. Hence, there is a finite limit to gradients that can be obtained with external gradient systems. In copending U.S. Provisional Application No. 60/098,021, means for maximizing radial gradients and methods for maximizing separation efficiency via a novel vessel design are disclosed. Such vessels can be used for practicing the methods described herein.
Another type of HGMS separator utilizes a ferromagnetic collection structure that is disposed within the test medium in order to 1) intensify an applied magnetic field and 2) produce a magnetic field gradient within the test medium. In one known type of internal HGMS system, fine steel wool or gauze is packed within a column that is situated adjacent to a magnet. The applied magnetic field is concentrated in the vicinity of the steel wires so that suspended magnetic particles will be attracted toward, and adhere to, the surfaces of the wires. The gradient produced on such wires is inversely proportional to the wire diameter whereas magnetic xe2x80x9creachxe2x80x9d decreases with diameter. Hence, very high gradients can be generated.
One drawback of internal gradient systems is that the use of steel wool, gauze material, or steel microbeads, may entrap non-magnetic components of the test medium by capillary action in the vicinity of intersecting wires or within interstices between intersecting wires. Various coating procedures have been applied to such internal gradient columns (U.S. Pat. Nos. 4,375,407 and 5,693,539), however, the large surface area in such systems still creates recovery issues via adsorption. Hence, internal gradient systems are not desirable, particularly when recovery of very low frequency captured entities is the goal of the separation. Further, they make automation difficult and costly.
On the other hand, cell separations using HGMS based approaches with external gradients provide a number of conveniences. First, simple laboratory tubes such as test tubes, centrifuge tubes, or even Vacutainers (used for blood collection) may be employed. When external gradients are of the kind where separated cells are effectively monolayered, as is the case with quadrupole/hexapole devices as described in U.S. Pat. No. 5,186,827 or the opposing dipole arrangement described in U.S. Pat. No. 5,466,574, washing of cells and subsequent manipulations are facilitated. Furthermore, recovery of the cells from tubes or similar containers is a simple and efficient process. This is particularly the case when compared to recoveries from high gradient columns. Such separation vessels also provide another important feature which is the ability to reduce volume of the original sample. For example, if a particular human blood cell subset, (e.g. magnetically labeled CD34+cells), is isolated from blood diluted 20% with buffer to reduce viscosity, a 15 ml conical test tube may be employed as the separation vessel in an appropriate quadrupole magnetic device. After appropriate washes and/or separations and resuspensions to remove non-bound cells, CD34+cells can very effectively be resuspended in a volume of 200 xcexcl. This can be accomplished, for example, by starting with 12 ml of solution (blood, ferrofluid and dilution buffer) in a 15 ml conical test tube, performing a separation, discarding the supernatant and subsequent wash supernatants and resuspending the recovered cells in 3 ml of appropriate cell buffer. A second separation is then performed which may include additional separation/wash steps (as might be necessary for doing labeling or staining reactions) and finally the isolated cells are easily resuspended in a final volume of 200 xcexcl. By reducing volume in this sequential fashion and employing a vortex mixer for resuspension, cells adhered to the tube above the resuspension volume are recovered into the reduced volume. When done carefully and rapidly in appropriately treated vessels, cell recovery is quite efficient (70-90%).
The efficiency with which magnetic separations can be done and the recovery and purity of magnetically labeled cells will depend on many factors. These include such considerations as: the numbers of cells being separated, the density of characteristic determinants present on such cells, the magnetic load per cell, the non-specific binding of the magnetic material (NSB), the technique employed, the nature of the vessel, the nature of the vessel surface, and the viscosity of the medium. If non-specific binding of a system is relatively constant, as is usually the case, then as the target population decreases so does the purity. For example, a system with 0.2% NSB that recovers 80% of a population which is at 0.25% in the original mixture will have a purity of 50%. Whereas if the initial population was at 1.0%, the purity would be 80%.
It is important to note that the smaller the population of a targeted cell, the more difficult it will be to magnetically label and recover. Furthermore, labeling and recovery are markedly dependent on the nature of the magnetic particle employed. As an example, large magnetic particles, such as Dynal beads, are too large to diffuse and effectively label cells in suspension through collisions created by mixing of the system. If a cell is in a population of 1 cell per ml of blood, or even less, as could be the case for tumor cells in very early cancers, then the probability of labeling target cells will be related to the number of magnetic particles added to the system and the length of time of mixing. Since mixing of cells with such particles for substantial periods of time will be deleterious, it becomes necessary to increase particle concentration as much as possible. There is, however, a limit to the quantity of magnetic particles that can be added. Instead of dealing with a rare cell mixed in with other blood components, one contends with a rare cell mixed in with large quantities of magnetic particles upon separation. The latter condition does not markedly improve the ability to enumerate such cells or examine them. Hence, the compromise is to limit the quantity of magnetic material and the mixing times, while enabling isolation of very rare target entities.
Another drawback to the use of large particles to isolate cells in rare frequencies (1 to 25-50 per ml of blood) is that large particles tend to cluster around cells in a cage-like fashion making them difficult to xe2x80x9cseexe2x80x9d or to analyze. Hence, the particles must be released before analysis, which clearly introduces other complications.
In theory, the use of colloidal magnetic particles in conjunction with high gradient magnetic separation appears to be the method of choice for separating a cell subset of interest from a mixed population of eukaryotic cells, particularly if the subset of interest comprises a small fraction of the entire population. With appropriate magnetic loading, sufficient force is exerted on a cell such that it could be isolated even in a media as viscous as that of moderately diluted whole blood. As noted, colloidal magnetic materials below about 200 nm will exhibit Brownian motion that markedly enhances their ability to collide with and magnetically label rare cells. This is demonstrated, for example, in U.S. Pat. No. 5,541,072 where results of very efficient tumor cell purging experiments are described employing 100 nm colloidal magnetic particles (ferrofluids). Just as important, colloidal materials at or below the size range noted do not generally interfere with viewing of cells. Cells so retrieved can be examined by flow cytometry or by microscopy employing visible or fluorescent techniques. Because of their diffusive properties, such materials, in contrast to large magnetic particles, readily xe2x80x9cfindxe2x80x9d and magnetically label rare events such as tumor cells in blood.
There is, however, a significant problem associated with the use of ferrofluid-like particles for cell separation in external field gradient systems which, for reasons given above, are the device designs of choice. Direct monoclonal antibody conjugates to sub-micron size magnetic particles of the type described by Owen or Molday, such as those produced by Miltenyi Biotec, do not have sufficient magnetic moment for use in cell selection methods employing the best available external magnetic gradient devices, such as the quadrupole or hexapole magnetic devices described in U.S. Pat. No. 5,186,827. When used for separations in moderately diluted whole blood, they are even less effective. Using similar materials which are substantially more magnetic, such as those described in U.S. Pat. No. 5,597,531 to Liberti and Pino, more promising results have been obtained. In model spiking experiments, it has been found that SKBR3 cells (breast tumor line), which have a relatively high epithelial cell adhesion molecule (EpCAM) determinant density, are efficiently separated from whole blood with direct conjugates of anti-EpCAM ferrofluids even at very low spiking densities (1-5 cells per ml of blood). On the other hand, PC3 cells (a prostate line) which have low EpCAM determinant density are separated with significantly lowered efficiency. Most likely this is a consequence of inadequate magnetic loading onto these low determinant density cells.
In light of the foregoing, the present inventors have appreciated a need for methods directed at increasing or enhancing the xe2x80x9cloadingxe2x80x9d of magnetic particles, whether large, small or colloidal, onto biological entities of interest. These methods may be used to advantage to isolate target substances or cells having low determinant density. Enhancing the efficiency of target bioentity isolation in turn faciliates subsequent biochemical and histochemical analysis of such entities.
It is the object of this invention to provide an efficient method for enhancing the interactions between specific binding pair members by systematically forcing collisions between the pair members present in a biological solution in a controlled and optimized fashion. This is accomplished by creating motion of one specific binding pair member relative to the other member such that increased numbers of collisions will occur. At the microscopic level, it appears that vigorous stirring of a solution may not always result in optimal numbers of collisions between members of a specific binding pair. For example, colloidal magnetic nanoparticles conjugated to monoclonal antibodies specific for a cell surface determinant, when mixed with target cells to be magnetically labeled, will label such cells much more efficiently and at a higher labeling density when the two entities are moved relative to each other. Thus, if cells are in suspension and the magnetic colloid is xe2x80x9cpulledxe2x80x9d through the cells, substantially greater numbers of nanoparticles are specifically bound to cells as compared to continued stirring, vortexing, and other means of mixing. Alternatively if the magnetic colloid is stable to centrifugation, then the cells may be moved through the colloid by centrifugation. This process also significantly increases the quantity of colloidal nanoparticles specifically bound to target cells. Hence the principle can be used in place of mixing or in addition to it.
There are many ways in which to induce motion of one entity relative to the other. In the examples provided below, magnetic gradients are used to translate particles through a suspension of cells; or centrifugation is used to move cells through gravitationally stable colloidal magnetic particles. Clearly in the latter case, neither component needs to be magnetic. For example, cells could be centrifuged through any colloid stable to the g-force of the process, such as centrifuging cells through colloidal gold.
In addition to ways for causing relative translation of the specific binding pair members mentioned above, charge or charge differential of one component relative to the other can be employed, as can any differential including magnetic or gravitational forces. One could envision an oscillating electrical field that would cause one entity to oscillate through the media relative to some other component. Similarly inertial forces may be employed
There are numerous applications where the methods of the invention are used to advantage, including but not limited to, immunoassay, cell separation, protein isolation for analytical use or for bioprocessing, bacteria capture, and nucleic acid manipulation. There are industrial processes where two entities must bind or collide to form a product or a product intermediate. This invention may be used to reduce the time of incubation or lower the temperature of the reaction, as temperature is frequently used to accelerate such reactions. The methods of the invention effectuate efficient specific binding pair member interactions desirable for the various applications set forth above. In the case of magnetic separations, bioentities having low determinant density are captured due to the increased magnetic loading of ligand specific particles made possible by this invention. Furthermore, the invention should provide enormous benefit by permitting the use of reduced concentrations of one of the specific binding pair members. If magnetic particles (or any other agent) can be brought into contact in some repeated fashion with a target entity associated with one specific binding pair member, then a lesser amount should be required to achieve the same level of labeling.
As used herein, the term xe2x80x9ctarget bioentitiesxe2x80x9d or xe2x80x9canalytexe2x80x9d refers to a wide variety of materials of biological or medical interest. Examples include hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemical substances, nucleic acid molecules, (RNA or DNA) and particulate analytes of biological origin, which include bioparticles such as cells, viruses, bacteria and the like. The term xe2x80x9cdeterminantxe2x80x9d when used in reference to any of the foregoing target bioentities, may be specifically bound by a biospecific ligand or a biospecific reagent, and refers to that portion of the target bioentity involved in, and responsible for, selective binding to a specific binding substance whose presence is required for selective binding to occur. In fundamental terms, determinants are molecular contact regions on target bioentities that are recognized by receptors in specific binding pair reactions. The term xe2x80x9cspecific binding pairxe2x80x9d as used herein includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin, and virus-receptor interactions. Various other determinant-specific binding substance combinations can conceivably be used in practicing the methods of this invention, as will be apparent to those skilled in the art. The term xe2x80x9cantibodyxe2x80x9d as used herein, includes immunoglobulins, monoclonal or polyclonal antibodies, immunoreactive immunoglobulin fragments, and single chain antibodies. Also contemplated for use in the invention are peptides, oligonucleotides, or a combination thereof which specifically recognize determinants with specificity similar to traditionally generated antibodies. The term xe2x80x9cdetectably labelxe2x80x9d is used herein to refer to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, is indicative of the presence of the target bioentity in the test sample. Representative examples of useful detectable labels, include, but are not limited to the following: molecules or ions directly or indirectly detectable based on light absorbance, fluorescence, reflectance, light scatter, phosphorescence, or luminescence properties; molecules or ions detectable by their radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. Included among the group of molecules indirectly detectable based on light absorbance or fluorescence, for example, are various enzymes which cause appropriate substrates to convert, e.g., from non-light absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules. The phrase xe2x80x9cto the substantial exclusion ofxe2x80x9d refers to the specificity of the binding reaction between the biospecific ligand or biospecific reagent and its corresponding target determinant. Biospecific ligands and reagents have specific binding activity for their target determinant yet may also exhibit a low level of non-specific binding to other sample components.